Malinda Salim

iTRAQ underestimation in simple and complex mixtures: "the good, the bad and the ugly".

The increasing popularity of iTRAQ for quantitative proteomics applications makes it necessary to evaluate its relevance, accuracy, and precision for biological interpretation. Here, we have assessed (a) the accuracy and precision of iTRAQ quantification in a controlled experimental setup, using low- and high-complexity protein mixtures; and (b) the potential pitfalls that hamper the applicability and attainable dynamic range of iTRAQ: isotopic contamination, background interference, and signal-to-noise ratio. Our data suggest greater dynamic crosstalk between interfering factors affecting underestimations, and that these interferences were largely scenario-specific, dependent on sample complexity. The good is the potential for iTRAQ to provide accurate quantification spanning 2 orders of magnitude. This potential is however limited by two factors. (1) The bad: the existence of isotopic impurities that can be corrected for; provided accurate isotopic factors are at ones disposal. (2) The ugly: we demonstrate here the interference of mixed MS/MS contribution occurring during precursor selection, an issue that is currently very difficult to minimize. In light of our results, we propose a list of advice for iTRAQ data analysis that could routinely ameliorate quantitative interpretation of proteomic data sets.

Minimising iTRAQ ratio compression through understanding LC-MS elution dependence and high-resolution HILIC fractionation.

Application of iTRAQ‐based workflows for protein profiling has become widespread. Concomitantly, the idiosyncratic limitations of iTRAQ, such as its tendency to underestimate quantifications, have been studied and recognised. This report shows that the influence of ratio compression and limiting transmission in iTRAQ MS/MS in high‐complexity mixtures (iTRAQ‐labelled lysates) can be partly alleviated using high‐resolution sample fractionation. Here, we also investigate in greater detail the dependency of iTRAQ quantification on the dynamics of online chromatography in low‐complexity mixtures (iTRAQ‐labelled standards). These findings will allow more efficient strategies to be designed for iTRAQ proteomics, alleviating iTRAQ underestimation and thus facilitating the detection of subtle abundance changes.

Studies of electroosmotic flow and the effects of protein adsorption in plasma-polymerized microchannel surfaces.

This paper presents a study of EOF properties of plasma‐polymerized microchannel surfaces and the effects of protein (fibrinogen and lysozyme) adsorption on the EOF behavior of the surface‐modified microchannels. Three plasma polymer surfaces, i.e. tetraglyme, acrylic acid and allylamine, are tested. Results indicate EOF suppression in all plasma‐coated channels compared with the uncoated glass microchannel surfaces. The EOF behaviors of the modified microchannels after exposure to protein solutions are also investigated and show that even low levels of protein adsorption can significantly influence EOF behavior, and in some cases, result in the reversal of flow. The results also highlight that EOF measurement can be used as a method for detecting the presence of proteins within microchannels at low surface coverage (<1 ng/cm2 on glass). Critically, the results illustrate that the non‐fouling tetraglyme plasma polymer is able to sustain EOF. Comparison of the plasma‐polymerized surfaces with conventionally grafted polyelectrolyte surfaces demonstrates the stabilities of the plasma polymer films, enabling multiple EOF runs over 3 days without deterioration in performance. The results of this study clearly demonstrate that plasma polymers enable the surface chemistry of microfluidic devices to be tailored for specific applications. Critically, the deposition of the non‐fouling tetraglyme coating enables stable EOF to be induced in the presence of protein.

Non-fouling microfluidic chip produced by radio frequency tetraglyme plasma deposition

This Technical Note presents the direct surface modification of a glass/PTFE hybrid microfluidic chip, via radio frequency glow discharge plasma polymerisation of tetraethlylene glycol dimethylether (tetraglyme), to produce hydrophilic, non-fouling, PEO-like surfaces. We use several techniques including X-ray photoelectron spectroscopy (XPS), direct enzyme-linked immunosorbent assays (ELISA) and immunofluorescent imaging to investigate the channel coatings. Our results indicate the successful deposition of a PEO-like coating onto microchannel surfaces that has both solution and shelf stability (>3 months) and is capable of preventing fibrinogen adsorption to the microchannel surfaces.

Amphiphilic designer nano-carriers for controlled release: from drug delivery to diagnostics

Vesicles formed by self-assembly of lipids and surfactants are increasingly recognised as carriers for drug delivery applications in disease targeting and many other biomedical-related areas, demonstrable by the growing number of significant publications. This manuscript reviews important facets of lipid-based vesicles as drug carriers and their surface modifications to achieve controlled release and selective cell targeting. We cover both the more commonly used ionic phospholipid vesicle carriers and the rapidly growing field of non-ionic vesicles/niosomes using self-assembly of uncharged amphiphilic molecules, which could be formed by using sugar surfactants or glycolipids, sorbitan esters, and polyoxyethylene alkyl ethers. Due to their lower cost, biodegradability, low-toxicity, low-immunogenicity and specific sugar-cell recognition, much attention would be devoted to glycolipid bio-surfactants as potential carriers for targeted delivery. Specifically, our review points to the design consideration of lipid and surfactant nano-carriers based on critical packing parameter, membrane curvature, and the effects of hydrophobic chain structures. We have also dedicated a section of this review to summarise some novel applications of various lipid liquid crystal phases in drug delivery, and how in turn these are related to chemical structures of the lipid entities. The final section of this review outlines the application of lipid vesicles as delivery agents for diagnostic imaging.

Characterization of fibrinogen adsorption onto glass microcapillary surfaces by ELISA

Adsorption of biomolecules onto microchannel surfaces remains a critical issue in microfluidic devices. This paper investigates the adsorption of fibrinogen on glass microcapillaries using an immunoassay method (ELISA) and X-ray photoelectron spectroscopy (XPS). Various adsorption conditions such as protein concentrations and incubation times, buffer pH, buffer ionic strengths and effects of flow are presented. ELISA is successfully demonstrated as a facile and robust technique to examine these phenomena. The highest adsorption level occurs near the isoelectric point of fibrinogen (pH 5.0) and low buffer ionic strengths (0-8 mM). Microchannel surface saturation was achieved at a fibrinogen solution concentration of approximately 50 microg ml(-1). Fibrinogen adsorption under flow was always higher than that seen in static systems. The importance of diffusion phenomena in microchannels on protein adsorption was demonstrated. ELISA experiments using fused silica and PEEK have also confirmed significant adsorption on these mass spectrometer transfer line materials.

Balancing robust quantification and identification for iTRAQ: Application of UHR‐ToF MS

iTRAQ reagents allow the simultaneous multiplex identification and quantification of a large number of proteins. Success depends on effective peptide fragmentation in order to generate both peptide sequence ions (higher mass region, 150–2200 m/z) and reporter ions (low mass region, 113–121 m/z) for protein identification and relative quantification, respectively. After collision‐induced dissociation, the key requirements to achieve a good balance between the high and low m/z ions are effective ion transmission and detection across the MS/MS mass range, since the ion transmission of the higher m/z range competes with that of the low m/z range. This study describes an analytical strategy for the implementation of iTRAQ on maXis UHR‐Qq‐ToF instruments, and discusses the impact of adjusting the MS/MS ion transmission parameters on the quality of the overall data sets. A technical discussion highlights a number of maXis‐specific parameters, their impact of quantification and identification, and their cross‐interactions.

Cubosome particles of a novel Guerbet branched chain glycolipid

ABSTRACT Cubic liquid crystalline nanoparticles (cubosomes) of bicontinuous nature with internal networks of water channels have received great interests in nanomedicine applications, particularly as potential vehicle for loading and release of therapeutic agents. These nanoparticles have been most commonly produced using monoolein and phytantriol. In this study, we explore the use of a Guerbet branched chain glycolipid, namely 2-hexyl-decyl-β-D glucopyranoside (β-Glc−OC10C6), as a new and alternative material for cubosomes production. The fully hydrated glycolipid assumes a reverse bicontinuous cubic liquid crystal phase of an Ia3d space group with lattice parameter of ca. 74 Å, as confirmed using a small-angle X-ray scattering. Dynamic light scattering and a conventional transmission electron microscopy were used to investigate the average size and morphology of the cubosomes. The effectiveness of Poloxamer 407 (stabiliser typically used in other cubosome systems against aggregations and particle coalescence) in providing steric stabilisation of the glycolipid cubosomes was assessed through visual assessment. GRAPHICAL ABSTRACT

Biomass derived xylose Guerbet surfactants: thermotropic and lyotropic properties from small-angle X-ray scattering

A series of novel branched-chain glycosides were synthesised from xylose, an aldopentose and Guerbet alcohols whose total number of carbon atoms ranges from C8–C24. The thermotropic and lyotropic phases of the highly pure Guerbet xylosides were investigated, using differential scanning calorimetry, optical polarising microscopy and small-angle X-ray scattering. In dry conditions, the shortest compound (total C8) exhibits a monotropic lamellar (Lα) phase while the C12 compound forms a rectangular columnar (Colr) phase at room temperature, a rarely reported phase in lipidic systems. The longer ones (C16, C20 and C24) adopt inverse hexagonal (HII) phases. Upon hydration, the Lα phase of the C8 compound remains, and while that of the C12 compound exhibits an inverse bicontinuous cubic phase of space group Ia3d in excess water. The C16 and C20 compounds remain in the HII phase upon hydrating, but the lattice parameters for the hydrated forms increase considerably. An inverse micellar Fd3m cubic phase is observed in the fully hydrated C24 compound. The amphiphilic nature of these compounds coupled with the ability to form inverse non-lamellar phases at room temperature makes them ideal candidates for a variety of applications such as controlled release drug-carriers.

Swelling of Bicontinuous Cubic Phases in Guerbet Glycolipid: Effects of Additives

Inverse bicontinuous cubic phases of lyotropic liquid crystal self-assembly have received much attention in biomedical, biosensing, and nanotechnology applications. An Ia3d bicontinuous cubic based on the gyroid G-surface can be formed by the Guerbet synthetic glucolipid 2-hexyl-decyl-β-d-glucopyranoside (β-Glc-OC6C10) in excess water. The small water channel diameter of this cubic phase could provide nanoscale constraints in encapsulation of large molecules and crystallization of membrane proteins, hence stresses the importance of water channel tuning ability. This work investigates the swelling behavior of lyotropic self-assembly of β-Glc-OC6C10 which could be controlled and modulated by different surfactants as a hydration-modulating agent. Our results demonstrate that addition of nonionic glycolipid octyl-β-d-glucopyranoside (β-Glc-OC8) at 20 and 25 mol % gives the largest attainable cubic water channel diameter of ca. 62 Å, and formation of coacervates which may be attributed to a sponge phase were seen at 20 mol % octyl-β-d-maltopyranoside (β-Mal-OC8). Swelling of the cubic water channel can also be attained in charged surfactant-doped systems dioctyl sodium sulfosuccinate (AOT) and hexadecyltrimethylammonium bromide (CTAB), of which phase transition occurred from cubic to a lamellar phase. Destabilization of the cubic phase to an inverse hexagonal phase was observed when a high amount of charged lecithin (LEC) and stearylamine (SA) was added to the lipid self-assembly.